Optical assembly and method for detection of light transmission

ABSTRACT

An optical assembly comprising a light source, at least one sample vessel and a detector, the at least one vessel being positioned in a light path or paths created between the source and the detector in manner to enable transmission of light through the vessel wherein the light source is adapted to provide a beam of substantially collimated light, the detector comprises a plurality of detector locations and the vessel comprises a wall and core of relative shape and dimensions adapted to contain a sample for detection and to define at least two spatially separated transmitted light paths, a first wall path which enters and exits the vessel walls only, spatially separated from a second core path which enters and exits the vessel walls and additionally the vessel core, wherein the spatially separated wall and core paths are coupled to individual detector locations on the detector, a module or clip-on device therefor, method for detection and uses thereof.

The present invention relates to an optical assembly comprising a samplevessel positioned in a direct light path between a light source and alight detector, in manner to enable transmission of light through thevessel; a method for detection of light transmission through samplecontained within the vessel; an apparatus comprising the assembly; moreparticularly an apparatus for sample analysis for example for highthroughput screening (HTS) or profiling or assays, such as enzymeassays; and uses thereof in the pharmaceutical, biomedical andbioscience, agrochemical, veterinary, materials and like fields, fordetection, analysis, characterisation and quantification or the like ofsamples contained in a vessel, and optionally further collectingseparated components thereof; in particular in combinatorial chemistry;in metabolomics, proteomics or genomics, assay and high throughputanalysis applications, typically high sensitivity analyses, separationand/or quantification studies and for sample separation for examplechromatography or electrophoresis, in particular column chromatography,capillary electrophoresis with real time or post separation analysis.

UV absorbance, fluorescence and mass spectrometry are key technologiesused in separation science for analysing species in samples. Aparticularly useful methodology is to look at a sample populationseparated by capillary electrophoresis with fluorophore labelling andfluorescence imaging, for quantification, and MS for characterisingmolecules of interest.

U.S. Pat. No. 5,582,705 discloses an apparatus and system for laserinduced fluorescence (LIF) detection in a multiplexed capillaryelectrophoresis system. A coherent beam incident on the capillary arrayand emitted fluorescent light are typically perpendicular to each otherin order to reduce background noise due to light scattering. Atransparent portion in each capillary wall defines a transparent pathextending through the array, perpendicular to the capillary. A 2D imagearray detector such as a charge-coupled device (CCD), preferably acharge-injection device (CID), is positioned to detect emission, and animaging lens interposed between the capillary array and the image arraydetector, to optically couple the pixels to the capillary. The imaginglens may be any lens capable of transforming an image onto the pixels ofthe image array detector, such as a camera lens or a condenser lens.Coupling is shown in FIG. 4, of U.S. Pat. No. 5,582,705 in which everysecond pixel is coupled to a sidewall of the capillary and every pixelin between is coupled to an interior portion.

Fluorescence detection is limited in its application since only alimited number of molecules are naturally fluorescent and many have tobe derivatised in reproducible and quantitative manner. Absorbancedetection therefore has the advantage of enabling detection of a widerrange of molecules. For example in enzyme assays, conducted inmicrotitre wells, techniques can be extended to absorbance detection ofchromophoric, UV and vis absorbing substrates consumed or produced in anassay, extending the range of assay to natural as well as syntheticsubstrates.

However a limitation of absorbance detection lies in the operablewavelength of detection. Absorbance detection is conducted on substratesin solution. However many common solvents absorb significant amounts oflight at wavelengths below ˜200 nm, and the resulting solvent absorptionsignal distorts and masks signals resulting from the substrate to bedetected. Accordingly absorbance detection is in practice limited todetection at wavelengths in excess of 190 nm, in the range UV-vis tonear infra-red (NIR).

Moreover a fundamental limitation of single point absorbance detectionis the impossibility of creating an image of the source at the detectionpoint on capillary that is brighter than the light source. In “A chargecoupled device array detector for single-wavelength and multi-wavelengthultraviolet absorbance in capillary electrophoresis”, Bergstrom andGoodall, Pokric and Allinson, Anal. Chem. 1999, 71, 4376-4384 disclosesoptical detection in capillary electrophoresis by means of absorbancedetection, illuminating a length of the capillary using a fibre opticbundle and using a charge coupled device (CCD) camera to image the fulllength of the illuminated zone. In this publication light from a fibreoptic bundle is focused by a sapphire rod through the capillary core anddetected on the opposite side of the capillary, by this means,increasing the target light area enabling more of the lamp output to beused and increasing the total light flux. In this case light emanatesfrom the capillary core, so all light detected is useful and thedivergent beam obtained is imaged on to the CCD.

Such a system becomes more complex once a parallel capillary array isintroduced in place of the single capillary. Optics to focus light onthe core of each capillary would be extremely complex and thereforeirradiating both the core and walls of each capillary becomes apractical consequence.

WO 01/18528 (Yeung et al) discloses a method for analysing multiplesamples simultaneously by absorption detection of samples in a planararray of multiple containers, whereby stray light from adjacentcontainers is eliminated by distancing the detection means from thearray, preferably at a distance greater than 10 times the diameter of acontainer, suitable 10-100 times the diameter for example at a distanceof 1-30 cm. Containers are preferably cylindrical capillary tubes asshown in the art. The array comprises a control container if the lightsource is unstable. It is stated that the cross section of the containerand thickness of the capillary wall are not critical. A flat field lenspreferably images the containers on to the detection means.

We have now found that further improvements in absorbance detectionassemblies enables increasing the total light flux through a capillaryor other sample vessel, by virtue of simplification of opticalcomponents, without unduly large separation of capillary and detectorwhich is undesirable and reduces light collection efficiencycompromising path length, and therefore light intensity. The improvedassembly is of particular advantage in detection in multiplexedcapillary arrays and enables imaging a large area of a capillary arraywithout the need for imaging optics. This is a significant advantage,especially when working in UV for which it is very difficult andexpensive to produce suitable optics. The assembly has benefits howeverin both single capillary and array detection, in particular enabling asimple and improved exposure referencing and acceptably lowintercapillary cross talk without the need for optics. In addition abenefit of the assembly of the invention is that it is suitable foroperation at short pathlengths, by virtue of the increased total lightflux through the core of the capillary or other sample vessel, and thisreduction in pathlength may lead to opportunities to conduct absorbancedetection at lower wavelengths, less than 190 nm, without encounteringimpracticably high levels of solvent absorption.

Accordingly there is provided in the broadest aspect of the invention anoptical assembly comprising a light source, at least one sample vesseland a detector, the at least one vessel being positioned in a light pathor paths created between the source and the detector in manner to enabletransmission of light through the vessel wherein the light source isadapted to provide a beam of substantially collimated light, thedetector comprises a plurality of detector locations and the vesselcomprises a wall and core of relative shape and dimensions adapted tocontain a sample for detection and to define at least two spatiallyseparated transmitted light paths, a first wall path which enters andexits the vessel walls only, spatially separated from a second core pathwhich enters and exits the vessel walls and additionally the vesselcore, wherein the spatially separated wall and core paths are coupled toindividual detector locations on the detector.

Preferably the detector is an array detector. Preferably the detector isadapted to detect and provide information on respective wall and corepath light transmission. Preferably the assembly is coupled to means fordisplaying information on respective wall and core path lighttransmission or for displaying referenced information on core path lighttransmission, referenced against wall path light transmission.

Preferably the assembly defines a central core path and two peripheralwall paths either side thereof or an annular wall path thereabout. Pathsmay overlap on emerging from the vessel and at greater separations fromthe vessel. Preferably the wall and core paths are coupled to detectorlocations at a vessel outer wall to detector separation or distance d atwhich the paths are spatially separated, preferably giving more than 90%separation of core and wall beam fluxes. The assembly may position thevessel in two or more separate light paths to generate two or more setsof spatially separated transmitted light paths coupled to two or moredetectors or detector zones.

Preferably the assembly is characterised by internal vessel dimension orpath length in the range of 3 μm to 20 mm, external vessel dimension inthe range 4 μm to 30 mm, refractive index of vessel wall in the range1.3-<1.6, vessel outer wall to detector separation d in the range 10 μmto >300 mm and is for use in detecting a sample comprising analyte insolvent having refractive index in the range 1.3 to in excess of 1.5.Reference herein to a sample is to vessel contents which may comprise asingle or multiple components. Multiple components may be present as ahomogeneous or heterogeneous mixture, and may undergo migration withtime ie may be a plurality of liquid phase components optionallyincluding a dissolved phase component; or may include one or a pluralityof analytes which it is desired to detect in one or a plurality ofsolvents or like bulk phase sample component, for example in the courseof a chemical reaction generating or consuming a species as analyte.

In a particular advantage, the apparatus of the invention enablesexposure referencing of a light beam traversing a core path of the atleast one sample vessel, by a light beam traversing a wall path of thesame sample vessel. The beams are spatially close, preferably adjacent,on the array detector, facilitating direct referencing as the ratio ofthe core beam to the wall beam. In a further advantage the two lightbeams are of neighbouring origin whereby core and wall beams have a highprobability of emanating from the same region in the light sourceeliminating the effects of light source fluctuations due to e.g.instability or spatial inhomogeneity. The assembly of the invention istherefore able to operate at the shot noise limit.

Preferably the light source comprises any active or passive lightsource, for example light may be generated at the source or it may betransmitted to the source and emanate therefrom, for example it may betransmitted by an optical fibre to the light source. Preferably thelight source comprises at least one wavelength of light that is absorbedby one or more absorbing species, the absorbance of which is to bedetected. For example the light source output may be coupled from afibre optic if desired for illumination from a remote light generator ormay be coupled from a point to line optical fibre for zone illumination.Coupling the output into a single optical fibre reduces noisecontributions caused by fluctuations in the spatial distribution of thelamp discharge.

Light may be of wavelength in the range 160 to 1200 nm, preferably 180or 190 to 1200 nm, corresponding to UV, UV-vis to near infra red (NIR),and is preferably in the range 180 to 700 nm corresponding to UV-vis. Itis a particular advantage that the present invention enables highsensitivity absorption detection at lower path length in the range 3-500μm and this allows operation at lower wavelengths in the range 160 to190 nm corresponding to UV which would be impractical for absorbancedetection of samples in some solvents by known techniques. Accordinglythe method is more solvent independent than normal HPLC orspectrophotometric methods.

A light source may be a point or line source adapted to illuminate asection through a compact vessel or elongate vessel. A light source maybe of single wavelength or multiple discrete wavelengths or wavelengthrange. A light source may be a tuneable light source, giving a tuneablesmooth output, a line output lamp giving a very intense output at onewavelength only, a spectrum light source giving a wavelength range alongthe length of a line light source or the like; and may be continuous intime, or pulsed. Sources with continuous outputs are preferable whenspectra are to be acquired over a wavelength range. Line sourcestypically provide more intense outputs at characteristic wavelengths,and are beneficial when the samples absorb at these wavelengths.Wavelength selection in the case of continuous wavelength arc lamps forexample, is suitably by known technique such as interference filterpositioned between the light source and collimating means or between thecollimating means and the sample vessel, preferably before thecollimating means. Alternatively a filter wheel or variable interferencefilter may be employed for sequential wavelength detection at multiplediscrete wavelengths, or a wavelength dispersing device may be employedin the form of a monochromator such as a grating or prism which may beeither fixed in position or continuously variable and spreads light intoa spectrum, giving a varied wavelength along the length of the capillaryor at right angles to the length of the capillary.

Preferably a light source comprises an iodine, zinc, cadmium or mercurylamp, or laser, as line sources; or a deuterium, xenon or tungsten lampas continuous output lamp; or a xenon lamp as a pulsed output lamp.

More preferably the light source comprises a deuterium arc lamp (for UVlight absorption) or a xenon arc lamp (for UV-vis light absorption); orcomprises a tungsten lamp, more preferably a filament lamp (for visiblelight absorption), and the like; most preferably a high output arc lampselected from deuterium, iodine, zinc, cadmium, mercury or xenon above;or the light source comprises a line source, preferably for the UV oneof the following: iodine, zinc at 214 nm, cadmium at 229 nm, or mercuryat 185, 254 or 365 nm; or the light source comprises a laser, forexample in the UV a laser such as a frequency quadrupled Nd:YAG at 266nm or Nd:YLF at 262 nm, or a He—Cd laser at 325 nm.

The light source may be expanded and recollimated by known means, forexample using cylindrical and spherical lens elements and the like,preferably using an elongate lens or cylindrical optical component, suchas a cylindrical fused silica lens or the like, to produce a collimatedbeam suitable for zone illumination of a sample vessel array.

The at least one sample vessel in the assembly of the invention maycomprise a cell or conduit which may be closed or open ended and closedor open based and topped, intended for static or dynamic sampledetection. The vessel may be aligned for light transmission in anysuitable plane through the vessel. Suitably light transmission isthrough a plane perpendicular to or containing the vessel ends or baseand top.

Preferably the sample vessel is a single cell or one of a plurality ofcells in an array, such as a rectangular or square array, for example ina microtitre plate, well plate or multi sample plate; or is a capillary,such as a microcapillary or microfabricated channel as known in the artof microfluidic transport and separation, more preferably is a singlecapillary or microchannel or one of a plurality of capillaries ormicrochannels in a parallel array.

A sample vessel array is aligned in a plane perpendicular to thecollimated light path whereby light passes through one vessel only. Inthe case that direction of illumination is in a plane containing thevessel base and top, light enters and exits each vessel through asidewall (wall path) or enters through the top and exits through thebase (core path), and in this case vessels may be aligned in a parallelor matrix array; in the case that illumination is through a planeperpendicular to the vessel ends, light enters and exits each vesselthrough a near side sidewall (wall path) or enters and exits through anearside sidewall, emerges into the core, enters and exits an opposingsidewall (core path), and in this case vessels may be aligned in aparallel array, ie only one vessel deep.

For 1:1 illumination:detection, the collimated light path has dimensionssubstantially corresponding to the dimensions of width and length of thedetector array and to a desired width and length of each sample vesselwhich it is desired to optically detect. Illumination:detectionmagnification is preferably in the range 0.8-1.5:1 in the case of arraysto avoid problems with spatial overlap of light from neighbouringvessels, and is in the range 0.5-2:1 for single vessels.

Light passing through the vessel walls and core is refracted on enteringand exiting the wall(s) and additionally on entering and/or exiting thecore. In the case of illumination through the length of a cell orcapillary, or through the cross-section of a straight walled cell orcapillary, emergent wall and core light paths maintain their respectiveorders, ie there is substantially no cross-over or convergence ofrespective paths, at least at a distance d from the vessel outer wall tothe detector.

In a particular embodiment of the invention the vessel of the assemblyis a microfabricated device providing square cross section capillaries,and is suitable for Fabry-Perot illumination with enhanced lightabsorption through multiple passages through the vessel. In this casethe walls of the vessel are coated with a reflective coating, within anabsorption zone, whereby light enters the vessel core in an illuminationzone adjacent the absorption zone, through a nearside side wall, at anangle less than 90 degrees to the wall, and at least a portion of lightis reflected at the opposing wall, with repeated internal reflectionsthroughout the absorption zone and finally emergence from the opposingwall at the end of the absorption zone. Light traversing a wall path maybe similarly reflected for ease of alignment but is preferably notreflected and exposure referencing is performed as normal.

In the case of illumination through the cross-section of a curved walledcell or capillary, for example a circular cross-section capillary,emergent wall light paths reverse their respective orders about acentral core light path, ie there is cross-over of respective wallpaths, within a distance d to the detector. In this case the opticalassembly of the invention is characterised by refraction patternsthrough the vessel in order to be able to spatially separate wall andcore light paths. Preferably the assembly is characterised by respectiveouter and inner diameter of a sample vessel, and by respectiverefractive indices of vessel walls and of sample, whereby the wall andcore paths are spatially separated as hereinbefore defined.

Preferably the refractive index of the vessel wall is greater than thatof the bulk phase of any sample comprised in the core. Preferablyrefractive index of the wall is in the range 1.34-1.59. Preferablyrefractive index of bulk phase sample comprised in the core is in therange 1.32-1.48 more preferably 1.32-1.38. The refractive index ofvessel wall may be modified by cladding or otherwise incorporatinghigher refractive index material in the vessel wall as a wall section,lens or the like.

Preferably the vessel outer wall and inner wall are additionally ofshape and dimension whereby light transmitted through the core isconvergent and forms a beam having an undeflected beam path, i.e. havinga beam path continuous with the incident collimated light. Light passingthrough the wall of the sample vessel only, entering the wall at oneoutside wall location and exiting at a second outside wall location maybe deflected or undeflected, preferably if deflected is divergent withrespect to the core light path such that two classes of light paths areformed which are spatially separated.

It will be appreciated that by virtue of using a collimated light sourceand knowing the refractive index of the sample vessel and any samplecontained therein the light path passing through any point of the vesselcan be predicted for any given shape and dimension of vessel outer walland inner wall, chosen such that collimated light passing through asquare or rectangular cross section vessel, normal and parallel torespective walls, emerges substantially unrefracted and parallel; andcollimated light passing through a curved or angular cross sectionvessel emerges uniformly divergent or convergent with a gradation inangle of refraction. This enables production of an emergent light pathof high symmetry and/or uniformity, which can be manipulated for imagingand exposure referencing as hereinbefore defined.

Preferably the at least one sample vessel has a cross-section in a planeincluding the transmission light path, which is square or rectangular,curved circular or angular or a combination thereof and is symmetricalor asymmetrical, preferably is symmetrical. The sample vessel moreovercomprises an outer and inner wall which may be of similar cross-sectionor shape or may be different, for example one may be circular and onesquare. The sample vessel wall through the cross-section may becontinuous or non-continuous, for example the vessel may be open orclosed and is preferably closed. A closed vessel may have a continuouswall through its cross-section or may comprise continuous base and sidewalls with a separate seal or lid. Preferably the outer wall is squareopen or closed or circular closed, and the inner wall is square or wellshaped open or closed or is essentially square with microfabricatedconvex or concave inner wall sections acting as lens faces, or concave,convex or prism shaped outer wall sections acting as lens faces.

Preferably at least one of the outer and inner walls of the samplevessel is of circular cross-section, whereby refraction and spatialseparation of core and wall beams is achieved, preferably outer andinner walls are of coaxial circular cross-section thereby defining anannular wall having outer and inner diameters such that refraction andspatial separation of core and wall beams is achieved.

It will be appreciated that sample vessel dimensions, refractive indicesand/or vessel to detector distance may be selected according to thenature of vessel wall material and shape and the sample to be containedwithin the core in order to achieve the desired refraction and spatialseparation. Accordingly the apparatus is defined by a relation of thefollowing properties which are shown here as a flow scheme rather thanas a mathematical relation:$\left. {\frac{i.d}{o.d.} + {r.i.({solvent})} + {r.i.({vessel})}}\Rightarrow\frac{d_{\min}}{o.d.}\Rightarrow d_{\min} \right.$where + infers a spatial relation which would be calculated usingsuitable ray tracing software to give values for vessel and assemblydimensions.

Specifically, knowing dimensions and refractive indices, ray tracing,for example using Zemax software, may be used to produce diagramsallowing relationships between dimensions such as dmin/o.d. to bededuced. A schematic diagram showing the cross section of a cylindricalvessel and the surface of the detector is shown in FIG. 5: symbols,given here in brackets, are for the inner diameter (i.d.), the outerdiameter (o.d.), and the outer wall to detector distance (d). Preferablyfor example for a sample vessel having circular outer and inner wallcross-section and constructed of quartz or silica, and for solvent ofrefractive index in the range 1.325-1.345 (encompassing for exampletypical reversed phase HPLC solvents methanol, water and acetonitrile),the minimum distance for spatial separation of the core and wall beamsusing collimated light incident on the vessel may be calculated fromvalues given in Table 1. For example, for a ratio i.d./o.d.=0.50, theminimum value of d/o.d. to achieve beam separation (defined as >90%separation of core and wall beam fluxes) is 0.5. For a capillary withactual dimensions corresponding to this criterion, e.g. i.d. 100 μm ando.d. 194 μm, the minimum value of d/o.d. is 0.5 and thus the minimumvalue of d is 100 μm.

The advantage of having as high an i.d/o.d as possible is to have a lowvalue of d/o.d., in order to minimise detector cross-talk betweenadjacent vessels.

Values of d/o.d. greater than the minimum are permissible, but thegreater the value, the more the constraints on how close the vessels canbe positioned.

There are situations in which no separation of the core and wall beamsis possible. For example, for a cylindrical vessel, this occurs in twogeneric cases. Firstly, if the refractive index of the material in thecore is too low, for example air in a silica or glass vessel (FIG. 6).Secondly, if the ratio i.d./o.d. is too low, for example water in asilica or glass vessel of i.d./o.d. ratio 0.2 (FIG. 8). The actualdimensions for the capillary in FIG. 8 are i.d. 75 μm and o.d. 364 μm, acapillary type widely used in capillary electrophoresis. FIG. 7 showsthat for the same i.d. but a smaller o.d., 194 μm, beam separation isreadily achieved.

Consideration of the optical properties of filled cylindrical vesselsshow that the constraints of refractive index and i.d./o.d. areinterdependent, emphasising that for each vessel and contents type, eachcase should be considered on its merits.

In the case where the refractive index of the vessel contents is higherthan that of the vessel walls, no beam separation is possible except atdistances unreasonably close to the vessel outer wall.

The vessel may be associated with additional optical components in theemergent light path, to focus or otherwise manipulate one or both of thespatially separated beam types.

The array may comprise fixed or variable spacers between each samplevessel for adjusting spacing of emergent beams B in a sequence forvessels 1 2 3 etc, as shown in Figure A, having wall w and core c, forexample wherein each beam corresponds to an array detection location:

-   B1w B1c B1w B2w B2c B2w B3w B3c B3w . . .

If desired, adjacent wall beams may be coincident:

-   B1w B1c B1w/B2w B2c B2w/B3w B3c B3w/ . . .

Other out of step arrangements are possible but may require more complexcoupling and reading of detector location.

Spacers may comprise elongate filters designed to screen all or part ofa wall light path, or a filter to screen part of the vessel wall therebyconfining the wall light paths, in both cases for minimisingintercapillary stray light and cross talk.

A spacer may therefore be of any suitable material for combined spacingand filtration, optionally also serving as support means for the atleast one sample vessel, and is of suitable opaque material such as forexample an opaque glass filled polymer.

Sample vessels may be comprised of any transparent polymer, glass,quartz, silica, e.g. fused silica or other material, preferably opticalgrade. Optical grade polymers include the classes of siloxanes such aspolydimethylsiloxane (PDMS), amorphous polycycloolefins based onnorbornene, and polymethylmethacrylate PMMA, polycarbonate, and othertransparent flexible polymers and fluoropolymers. Flexible transparentpolymers are commercially available as transparent flexible tubing suchas polyetherimide such as Ultem, polypropylene, fluoropolymers includingthe transparent PCTFE, ECTFE, ETFE, PTFE, PFA, FEP or PVDF resins,preferably the fluoropolymer Tygon chemfluor 367 and the like.

The vessel wall may be transparent in part or whole and is preferably ofthe same material throughout.

A capillary as hereinbefore defined may comprise any extruded capillaryor microfabricated capillary or channel or other parallel elongateclosed conduit means. A capillary may be flexible or rigid and may bestraight or curved along all or part of its length.

Capillary arrays and microfabricated channel arrays are commerciallyavailable. Capillary arrays are typically manufactured by drawing orextrusion, creating a void circular cross-section core of highuniformity dimensions. Microfabricated arrays are typically manufacturedby injection moulding from a tool, creating a void square or rectangularcross-section channel, which is then optionally sealed with a coverlayer, and offers the advantage of absolute reproducibility of highlyprecise channels, and freedom of channel design.

A sample vessel may comprise part of an array of any number of vessels,commercially available arrays include 8 to 1536 vessels, preferably 8 to520 capillaries or 96 to 1536 wells, for example 8, 12, 16, 24, 32, 48or 96 capillaries or 96, 384 or 1536 wells.

The vessel may comprise part of an array of a greater number of vesselswhich may be available in the future.

Micro fabricated arrays fabricated in layered planar manner,constructing interfacing elements of a device in sequential planes, aregaining acceptance and widespread use. They have numerous advantages inaccuracy, reproducibility, ease and flexibility of manufacture.

Vessels may have any desired separation, suitably are fused or adjacentor are spaced apart. A lower separation increases packing density but ahigher separation reduces intervessel interference, in particularoptical interference. It is a particular advantage of the invention thatintervessel spacing may be used. This is not possible in the embodimentof Yeung et al above, for example which needs minimal vessel separationto minimise stray light.

The vessel may be of suitable length and depth according to the desireduse. Preferably for capillaries or channels length is in the range 2cm-2 m for example in the range 2.5-6 cm for microfabricated channels or6-100 cm length for capillaries, more preferably 10 to 60 cm. Greaterlength allows application of higher voltage in capillary electrophoresisand improved separation. For microtitre plate well, preferableinter-vessel centre to centre spacings are compatible with standardmicrotitre plate formats, such as 2.25, 4.5 and 9 mm.

Preferably a vessel is of 3 μm to 20 mm in depth or internal diameter,and 4 μm to 30 mm in external diameter, more preferably 9 μm-3 mm indepth or internal diameter and 10 μm to 3.5 mm in external diameter,more preferably 10-380 μm, most preferably less than or equal to 200 μmin depth or internal diameter or external diameter. A vessel wall isimportantly of suitable thickness for the optical properties of theinvention. This is particularly relevant for vessels of small internaldiameters. Accordingly vessel internal diameter or “bore” may beselected and vessel wall material selected, and suitable outer diameterdetermined accordingly to give the required optical properties. Forsilica capillaries with internal diameter (hereinafter i.d.) in therange 20 to 250 μm, for example 20, 25, 50, 75 or 100 μm, externaldiameters (hereinafter o.d.) is preferably in the range 100 to 380 μm,for example 150, 200 or 360 μm. For example a vessel may be of 75-100 μmi.d. and 194 μm o.d. or 180 μm i.d. and 364 μm o.d. It is important tonote that for a vessel with 75 μm i.d. and 364 μm o.d., as widely usedin capillary electrophoresis, no separation of core and wall beams ispossible when working with aqueous solutions. For silica capillaries,thickness is suitably in the range 30-500 μm, for example 30-150 μm:vessels of low i.d. preferably have low wall thickness, to give aminimum i.d. to o.d. ratio of 2-4 for example 0.25.

When working with larger diameter reaction vessels, similar constraintsapply, but normally the i.d./o.d. ratio falls in the range givingsuitable beam separation. For example, a glass reaction vessel having ani.d. of 20 mm and an o.d. of 30 mm has an i.d./o.d. ratio equal to 0.67,which permits beam separation with all common solvents (see Table 1).For a polycarbonate reaction vessel and water as solvent, for an i.d. of0.33 cm, an o.d. of 1.0 cm would be suitable, but an o.d. of 1.5 cmwould be unsuitable. For a representative fluoropolymer tubing, Tygonchemfluor 367, with i.d. 1/16″ (1.6 mm) and o.d. ⅛″ (3.2 mm), thei.d./o.d. ratio is 0.50 and beam separation with solvent water isachieved for all values of the ratio d/o.d. greater than 0.4, i.e. d>1/20″ (1.3 mm). However, when working with hexane as a solvent in suchtubing, the refractive index of the vessel contents is greater than thatof the walls (values of refractive indices are given in footnote toTable 1), and no beam separation is possible other than at distancesunreasonably close to the vessel outer wall.

The sample vessel may be void or packed, for example may comprisestationary phase or may be coated or otherwise configured with suitablematerials as known in the art for example in HPLC. Suitably packing ifpresent is of corresponding refractive index to bulk phase sample or tosolvent comprised in sample to be detected.

Preferably the vessel comprises internals and components characteristicof columns or capillaries present in pressure driven or electricaldriven separations including HPLC (for pressure driven) or capillaryelectrochromatography (CEC) (electrically driven equivalent of HPLC,separating by binding or partition coefficient, using eg hydrophobicstationary phases), micellar electrokinetic chromatography, andcapillary electrophoresis (CE) including the focusing and concentratingtechniques of isoelectric focusing (IEF separating by isoelectric pointindependent of size and shape of molecules), isotachophoresis (ITP),capillary zone electrophoresis (CZE separating by charge to size ratio)and dynamic field gradient focussing (DFGF, where a constanthydrodynamic force is opposed by a gradient in the electric field, whichallows charged molecules to focus in order of their apparentelectrophoretic mobilities and selective concentration of analytes e.g.proteins—see “Digitally controlled electrophoretic focusing” Huang andCornelius, Anal. Chem., 1999, 71, 1628-1632).

The separation between a sample vessel and the detector, d, is suitablysuch as to facilitate coupling of spatially separated light paths to thedetector locations. Suitably the separation is a function of the vesselo.d. and of the degree of separation of light paths, and is preferablygiven by the ratio d/o.d. is less than 10, for example is in the range0.5-5, preferably 0.5-1. In a particular advantage the optical assemblyof the invention allows an extremely compact structure when working withcapillaries in which separation d is of the order of 50-360 μm, forexample 200 μm. This is particularly advantageous since the lesser thevalue d the more compact and robust is the assembly and the more intenseare the transmitted light beams at the detector. The separation d may beadapted to couple spatially separated light paths in sequence ashereinbefore defined, with or without intervening optical components andpreferably without intervening optical components.

There are a number of commercially available array detecting systems,including for example the commonly used photodiodes and more recentlyavailable charge coupled devices (CCDs) and active pixel sensors (APSs)eg complementary metal oxide semiconductor (CMOS).

Preferably therefore an array detector according to the inventioncomprises a solid state sensing device, more preferably a CCD, CID or aCMOS APS.

A detection zone may be of any desired area size and is suitably of zonedimensions substantially equal to the vessel or array cross section in aplane perpendicular to the light path, corresponding to one to oneimage. A detection zone may comprise one or a plurality of arraydetectors.

Smaller zone size increases noise and reduces sensitivity yet can beprovided at lower cost. Preferably the apparatus comprises lowest noiseand highest sensitivity CCDs in a relatively small area device, such asa 1024×256 pixel CCD (MAT CCD30-11).

A CCD for use in the apparatus of the invention may have 22 to 5000 ormore pixels in either dimension, preferably 256, 512, 770, 1152, 2048 or4096 pixels, of pixel size 7 to 35 μm, preferably 20 to 30 μm, morepreferably 22 to 30 μm. Preferably the CCD comprises 3 to 28, forexample 10 pixels per capillary or 30 to 2500 for example 100 pixels perwell. Pixels outside the imaging area are preferably not digitised toreduce readout time.

Commercially available CCDs may include a stud, for protection of theCCD surface which is usually recessed in the CCD support package and toconserve image quality. Preferably the stud comprises a coating toabsorb incident light and reemit at a different wavelength, to convertUV to visible light, to allow detection by the CCD. A coating is forexample a phosphor coating. The phosphor coating may be applied directlyto the stud or to a cover slip interleaved between the stud andcapillary, facilitating changing phosphor as needed, by replacing thecover slip without need to replace the stud.

Each camera is interfaced to control means, preferably a processingcontrol means providing a suitable pixel readout rate, suitably of theorder of MHz, preferably greater than 4 or 5 MHz.

Preferably the processing means is programmed for selection of on-chipcharge binning procedures, to increase signal current (photoelectronsper second) and selection of area of interest for read out. Additionallythe processing means controls camera readout and collects, stores andanalyses data.

In the case of an assembly comprising a conduit sample vessel intendedfor dynamic sample detection, preferably the apparatus operates withreal-time signal processing for optimum peak detection andparameterisation/characterisation, and potential for automatic systemmanagement including closed-loop feedback control of the apparatus andsystems. For example, feedback could include stopping or slowing theflow following initial observation in the detection window, to allowsample to reside in the detector window and give longer times for dataacquisition and enhanced signal to noise or to facilitate detectionalong the length of a vessel.

Flow velocity may be controlled by adjustment of electric field (as incapillary electrophoresis) or by adjustment of pressure (as in liquidchromatography), or by a combination of the two. For flow driven bycentrifugal forces, flow velocity control is carried out by adjustmentof the angular velocity.

Suitably the assembly comprises means to detect presence of sample in adetection window, and feedback control means for turning off the voltagewhen it is detected as having migrated to the centre of the window (seeFIG. 13). Feedback control means suitably includes means for monitoringthe subsequent broadening of the sample peak due to diffusion as afunction of time, and for analysis of the change in peak variance withtime, and determining the diffusion coefficient, enabling thehydrodynamic radius of the analyte to be deduced, and means forcombining with the mobility, obtained from the time taken to reach thewindow under a given field strength, enabling the charge on the analyteto be deduced. The control means is able to operate with any startingpeak shape, since convolution of the peak with a Gaussian enables theGaussian component of the peak broadening to be extracted. Data qualityobtained with the assembly of the invention is such that diffusioncoefficients could be measured with good precision in less than 3minutes. This assembly could be used to measure diffusion coefficients,size and charges of both small molecules and large molecules, includingproteins, DNA, and polysaccharides, and offer a simpler alternative toanalytical ultracentrifuge methods.

Alternatively closed loop feedback could include means such as peakrecognition and decision making software for instructing a switchingvalve and controlling switching times to enable fraction collection, orto direct a fraction to a mass spectrometer, e.g. via an electrosprayinterface, or to a NMR spectrometer. Means for observing the flow streamboth before and after the valve enables the quality of analyte switchingand efficiency of the switching means to be monitored. A schematicexample of closed-loop feedback control is given in FIG. 14. Preferablythe assembly positions the capillary in two separate light paths and togenerate two sets of spatially separated transmitted light paths coupledto two detectors or detector zones with the switching valve therebetweenwhereby after the appropriate switch the eluent is monitored again in asecond pass of the detector, so that the efficiency of the switchingprocess can be monitored. An alternative is to have more of the outputsof the switching valve passing the detector for visualisation.

Closed loop feedback may also include controlling velocity of samples inmultiple vessels, for example in a capillary array, to co-ordinate exittimes, in sequence or, for combining samples from different capillariesinto a common exit means, simultaneously. Velocity may be controlled bycontrolling voltage or pressure, for example pumping flow rate, and maybe operated with switching devices. Velocity may enable stopping samplesflow at desired time to coincide with an event which is to be performedon the sample or is to take place in the sample. Controlled exit may befor subsequent fraction collection or analysis, for example forinterfacing the output of a capillary bundle to a mass spectrometer byelectro-spray, or to an NMR spectrometer or the like.

Preferably controlled loop feedback is provided by creating a potentialdifference across the length of the capillary and/or varying the appliedvoltage to control migration of sample, including slowing or stopping toimprove signal/noise ratio and including timing sample migration toachieve a desired time of arrival of sample at a point to switch into asample collection means such as mass spectrometer. The system mayoperate with a pressure difference and pumping, electrical field driver,column switching or the like. Preferably therefore a capillary compriseselectrodes, valves or pressure regulators at the ends thereof or alongthe length thereof about the detection zone or each detection zonetogether with circuitry to control electrodes or to facilitate pumpingcontrol. In the case of voltage controlled migration, preferably asample vessel is connected to a buffer supply and electric field isarranged such that buffer supply and sample introduction is at the highpotential end of the capillary, where high may be either positive ornegative with respect to ground.

In a further aspect of the invention there is provided an opticalassembly module for use with a column or capillary separating device asknown in the art, wherein the vessel is a capillary or column comprisinginterfacing means at one end for inserting into the outlet of a columnor capillary separating device and optionally comprising interfacingmeans enabling insertion into the inlet of an analysing means at theother end or comprising interface means at both ends for insertion alongthe length of a capillary or column. A module for insertion along thelength of a column or capillary typically has a column or capillarysection of matching i.d and o.d as the separating device or interfacingmeans allowing a smooth flow transition.

In a further aspect of the invention there is provided an opticalassembly clip-on device adapted to locate about a section of a capillaryor column which is of suitable i.d, o.d and refractive index ashereinbefore defined comprising means to locate about the capillary orcolumn of a separation device. Optionally the capillary or column hasits outlet inserted into the mass spectrometer, in which case theclip-on is located as close to the mass spectrometer as possible. In thecase of a clip-on, the capillary or column of the separation device maybe stripped of any surface coating to facilitate the operation of themethod of the invention, whereby the stripped capillary or column, suchas a capillary electrophoresis or microbore HPLC section, provides thesample vessel of the assembly.

In a further aspect of the invention there is provided an apparatus forchemical reaction or synthesis and analysis or for sample separation ortransport wherein the apparatus comprises the optical assembly ashereinbefore defined. The chemical reaction vessel itself could becylindrical and the reaction monitored in batch flow mode as a functionof time, and feedback control used to halt reaction—e.g by admixture ofa quenching reagent, by change of temperature, or by exporting thevessel contents. Alternatively the reaction vessel could be tubular andused in continuous flow mode. The apparatus may comprise a plurality ofmeans for introducing one or more reagents to the at least one samplevessel together with means for regulating reaction conditions to form adesired chemical synthetic reaction within the at least one samplevessel; or may include means for inlet and outlet of sample in dynamicfashion into the at least one sample vessel together with means forapplying a pressure difference or potential difference across the endsof the vessel and means for supplying separation mediums such as bufferin order to perform separations within the at least one sample vessels:or may include a separation means such as a chromatography column asknown in the art having outlet interfaced with an inlet end of the atleast one sample vessel as hereinbefore defined for dynamic analysis ofseparated sample.

In a further aspect of the invention there is provided a method fordetection of light transmitted through at least one sample containedwithin the core of at least one sample vessel of an optical assembly ashereinbefore defined, comprising illuminating the vessel with asubstantially collimated light source or sources and detectingtransmitted light in a detector, wherein transmitted light is spatiallyseparated into at least two light paths, a wall path which has passedthrough the vessel walls only, spatially separated from a core pathwhich has passed through the walls and core, wherein the spatiallyseparated light beams are coupled to individual detection locations onthe array detector. Suitably the method comprises any features orembodiments corresponding to apparatus features and embodiments outlinedabove.

Preferably the method is a method for detecting light from samples insample analysis for example for high throughput screening (HTS) orprofiling or assays, such as enzyme assays; and uses thereof in thepharmaceutical, biomedical and bioscience, healthcare, agrochemical,veterinary, industrial, environmental or materials or like fields, fordetection, analysis, characterisation and quantification or the like ofsamples contained in a vessel, and optionally further collectingseparated components thereof; in particular in combinatorial chemistry;in metabolomics, proteomics or genomics, assay and high throughputanalysis applications, typically high sensitivity analyses, separationand/or quantification studies and for sample separation for examplechromatography or electrophoresis, in particular column chromatography,capillary electrophoresis with real time or post separation analysis, inhospital or surgery blood tests and other assays, in industrial qualitycontrol, in environmental pesticide level monitoring and the like.

The method may be a method for detecting light from a static or dynamicsample. Typically a static sample is contained in a closed ended samplevessel as hereinbefore defined, more typically in a vessel in the formof a cell or well which may be one of a plurality of vessels for examplein a microtitre plate or a well plate or sample plate as hereinbeforedefined. Samples derived from enzyme assay or other multiple sampleanalysis may be in this form for detection.

In one embodiment the method is a method for single capillary ormulticapillary absorbance detection, comprising drawing solvent thensample into the capillary(s), by capillary action, pressure, pipettingor the like, conducting detection of difference in transmittance ofsample and solvent, and disposing of or blowing sample back to thesource. This method is useful in bioscience for making absorbancemeasurements on small sample volumes of microlitres or less. Enzymeassays may be conducted with one or two streams entering the vessel anddetecting a time dependent change, normally in profiling, for enzymeassay.

Typically dynamic samples are electrically or pressure driven and arepresent in an open ended conduit type vessel as hereinbefore defined,such as a capillary having inlet and outlet ends for flow of samplethrough the capillary, the capillary may be a single capillary or partof a capillary array as hereinbefore defined. Samples derived from highthroughput screening or from the separation or transport techniques maybe provided in this manner for detection, of which samples derived fromseparation techniques may be provided for separation within thecapillary with simultaneous detection of light transmission, or may beseparated in a separation method such as column chromatography, and theseparated sample flow from the column coupled directly into a capillaryfor detection of light transmission.

Preferably the method for detecting a dynamic sample is a method forcolumn or capillary separation as known in the art, suitably selectedfrom pressure driven or electrical driven separations including HPLC(for pressure driven) or capillary electrochromatography (CEC)(electrically driven equivalent of HPLC, separating by binding orpartition coefficient, using eg hydrophobic stationary phases), micellarelectrokinetic chromatography, and capillary electrophoresis (CE)including the focusing and concentrating techniques of isoelectricfocusing (IEF separating by isoelectric point independent of size andshape of molecules), isotachophoresis (ITP), capillary zoneelectrophoresis (CZE separating by charge to size ratio) and dynamicfield gradient focussing (DFGF, where a constant hydrodynamic force isopposed by a gradient in the electric field, which allows chargedmolecules to focus in order of their apparent electrophoretic mobilitiesand selective concentration of analytes e.g. proteins—see “Digitallycontrolled electrophoretic focusing” Huang and Cornelius, Anal. Chem.,1999, 71, 1628-1632). Other electrically driven techniques are known ormay be developed in future.

A method which is a method for electrophoretic separation of moleculesis carried out with an assembly, module or clip-on in a capillary orchannel as hereinbefore defined which is connected to a buffer supply.An electric field in the range of kilovolts is applied across both endsof the capillary or channel to cause the molecules to migrate. Samplesare typically introduced at a high potential end and, under theinfluence of the electric field, move toward a low potential end of thechannel. Absorbance analysis may be conducted along the length of thecapillary or channel or near the outlet allowing observing an entireprocess taking place in the length of the capillary or channel or theresult thereof.

Another use of dynamic detection is in measuring refractive indexchange. By analysis of the illumination pattern of the core beam at highspatial resolution perpendicular to the capillary axis, the detector hasthe ability to monitor refractive index change in the solvent in thecapillary. This would allow distinction between two solvents, and byextension the ability to directly monitor the mobile phase compositionduring a gradient elution separation in dual solvent mixtures. Ifnecessary, magnification in the direction perpendicular to the capillaryaxis could be used to increase the resolution in terms of mobile phasecomposition. This would be of benefit in HPLC. Since solvent refractiveindex may be temperature dependent, application of a heat pulseproviding a temperature rise up-stream of the detector could be used todetermine the mobile phase velocity in capillary HPLC.

Another use of multicapillary absorbance detection is for rapidmeasurement of pK_(a). Here aliquots of the sample solution are mixedinto a set of buffer solutions covering a range of pH values, typicallyspanning the range 2-12, and all mixtures are then drawn up intoseparate capillaries in the sequence buffer then mixture of buffer plussample. By taking measurements at one or more wavelengths where acid andbase forms have different absorbance, results from all capillaries ofabsorbance as a function of pH may be processed to determine pK_(a)values. Using suitable non- or weakly-absorbing inorganic buffers, thisis applicable for organic or biological compounds containing all commontitratable functional groups, including carboxyl and amine groups, andat concentrations down to 100 micromolar. This is of benefit in forexample the pharmaceutical industry and for high throughput screening.

The invention has particular use in relation to samples of smallmolecules of MW of the order 15-500, but may also be used in relation tosamples of larger molecules such as polymers, proteins, DNA and otherbiomolecules of MW of the order 500 to 106. The invention is ofparticular advantage in one embodiment due to its small size. This maybe employed to advantage in hospitals and the like for sample analysis,without the need for clinical biochemical laboratories, for example as acolour assay. Blood tests may be conducted on blood samples which arecurrently spun to separate red blood cells and plasma in a laboratory.The assembly of the invention may be used to detect red blood andplasma, either before or after centrifugation, for example by adding areagent to the plasma region and observing the reaction.

In an alternative embodiment multiple wavelength detection allowsdetection at a range of wavelengths along the length of a capillary orchannel.

Preferably the method is a method enabling or allowing sometransformation or event to occur in a vessel and imaging the event.Imaging may be conducted at the end of the transformation or event orthroughout. Imaging throughout an event requires timing to coincide withmigration of sample in the vessel, preferably using controlled loopfeedback as hereinbefore defined to stop the sample migration in thevessel at desired location or interval for imaging.

For example the method may be a method for observing a chemical reactionfor biomedical, bioscience, healthcare, agrochemical, veterinary,pharmaceutical, industrial or environmental purpose, wherein multipledetection may be performed on the inlet and outlet and in the waste toensure that all of the reaction product has been collected. The reactionmay be conducted in the vessel in the form of capillary which may becurved, allowing ease of detection of multiple positions using a commonlight source and detector.

In an alternative embodiment the method comprises detecting sample frompharmaceutical studies, in the form of analyte in solvent such asdimethylsulfoxide (DMSO), stored in microtitre plates, as is common inpharmaceutical practice. The method of the invention may be performed onsamples either in the microplate array in situ, as a vessel array ashereinbefore defined or following drawing up portions into one or morecapillaries, as a single capillary or capillary array. Use of a shortpath length of less than 9 mm, preferably of the order of 300 μm orless, allows UV absorbance detection without total absorption of lightby the DMSO solvent.

In an alternative embodiment the method is a method for measuringphysico-chemical properties for example partition coefficients,comprising placing sample of analyte in a first solvent in the vessel,together with a second immiscible solvent, for example water and octanolrespectively, and observing the analyte moving between the twoimmiscible phases. In a particular advantage the method of the inventionalso enables imaging of the solvent phases whereby their interface isalso visible.

In an alternative embodiment the method may be a method for detection oflight from a plurality of sample vessels for high throughput analysis ofdifferent samples in each vessel, for example for comparative analysisthereof or may be a high loading method for detecting in each vessel thesame samples from a high volume, high flow or otherwise bulk upstreamprocess, intended for combining a desired component from each sample. Ahigh loading method may comprise introducing the output from a singleHPLC for example having flow rate exceeding that possible in a singlecapillary, but suitable for introducing into a capillary array which maythen function effectively as a single detector. For example the methodmay comprise detecting analytes present in sample in or exiting amicrobore liquid chromatography column and the column may be of diameterin the micron range up to millimetre range. High loading in a pluralityor array of vessels achieves higher resolution in separation andpotentially higher sensitivity in detection than operation on a largescale in a single vessel from a single large bore separation channel.Moreover a shorter path length is possible in an array of capillariesthan is possible in a standard HPLC cell and this allows operation at alower wavelength as hereinbefore defined.

A method for detection in DFGF or multiple detection method preferablyemploys feedback control as hereinbefore defined.

Preferably the method comprises illuminating the at least one samplevessel with collimated light comprising a single or a plurality ofwavelengths selected in the range as hereinbefore defined. In aparticular advantage illumination is with a collimated light source of asingle wavelength at any given time, thereby simplifying readout ofoptical detection results.

A sample as hereinbefore defined may comprise any sample of one or moresmall or large molecules present in liquid or gel phase suitably insolution with liquid phase solvent or cosolvent such as an inert or nonreactive liquid. Solvent or gel may have refractive index in the rangeas hereinbefore defined. Suitably however these samples arecharacterised by a refractive index of lower than but of similar orderto that of the sample vessel walls, whereby the sample provides agenerally convergent light path. Preferably therefore the samplecomprises a solution or a suspension of molecules to be detected in asolution or suspending medium selected from water, alcohols,acetonitrile, hexane, dichloromethane, acetone, DMSO and other commonsolvents and cosolvents and mixtures thereof; or the sample may beprovided in the form of a gel for example uncrosslinked polymersolutions such as cellulose derivatives (e.g. hydroxypropyl cellulose)and synthetic polymers (e.g. polyethylene oxide).

Preferably the method of the invention comprises selecting a sample foranalysis, determining individual wavelengths at which absorption bydesired sample components is strongest, checking refractive index of thesample in order to select a suitable sample vessel which when containingthe sample and when illuminated will generate spatially separated beamsas hereinbefore defined or selecting a suitable combination of opticalcomponents, filters and the like and a suitable vessel to detect anarray separation to couple spatially separated beams to independentlocations on the detector array.

Sample may be introduced into the at least one sample vessel ashereinbefore defined in known manner, for example by injection, loopinjection, pipette, hydrostatic, electrokinetic or like injectiontechniques and may be removed from the vessel in known manner such asinjection, electrospray or other interface for discard or to a furthervessel for storage or to a down stream identification means such as massspectrometer.

Detected light is coupled to individual detection locations on the arraydetector as hereinbefore defined. The at least one sample vessel ashereinbefore defined is coupled to a plurality of detector locations inmanner that core detector locations correspond to the core light pathfrom the at least one vessel and peripheral detector locationscorrespond to the peripheral wall point(s) from the vessel. Preferablythe method comprises imaging the transmitted light detected by thedetection means, for example in the form of a CCD image as known in theart. Preferably the method also comprises referencing the light detectedby the detection means by means of exposure referencing wherein theratio of the core beam intensity to the wall beam intensity gives avalue for the sample intensity at each location with elimination ofexcess or flicker noise due to light source fluctuation.

Accordingly therefore the method comprises coupling the at least onesample vessel to at least three detector locations, preferably 3 to 25detector locations for a sample vessel of the order of microns diameterwherein the locations may be apportioned for example 1:1:1-5:15:5 or8:9:8 depending on the relative wall and core beam width; and for asample vessel of the order of cm in an amount of 30 to 2500 pixels persample vessel for example in the ratio 5:20:5 or 10:10:10 to500:1500:500 or 800:900:800 depending on the relative wall and core beamwidth. The method may include pixel summing or the like for enhancedsignal strength and noise reduction as known in the art.

Preferably the method includes subsequently measuring the amount ofabsorption of light by species in the sample vessel which indicates theamount of absorbing species in manner as known in the art and comprisingmeasuring the intensity of light in the absence and presence of thesample. In a particular advantage measurement according to the inventionis simply by measuring intensity of light in a wall beam and a corebeam. The logarithm of the ratio, taken in conjunction with valuesmeasured in the absence of sample, provides the absorbance according tothe Beer-Lambert Law.

Absorbance may be imaged, for example as a CCD snapshot. Preferablyhowever the method is a method for snapshot detection whereby detectionis recorded in a plurality of finite exposures, for example fiveexposures per second, and exposures are super imposed, directly in thecase of a static example or with time displacement in the case of adynamic sample. Accordingly an individual image reveals limitedinformation and therefore the detection array preferably provides rawdata which is compiled and converted to graphical display for example inthe form of an electropherogram.

In a further aspect of the invention there is provided the use of theoptical assembly, method and apparatus as hereinbefore defined in sampleanalysis for example for high throughput screening (HTS), for example inan array as hereinbefore defined, or profiling or assays, such as enzymeassays; and uses thereof in the pharmaceutical, biomedical andbioscience, healthcare, agrochemical, veterinary, materials, industrial,environmental, and like fields, for detection, analysis,characterisation and quantification or the like of samples contained ina vessel, and optionally further collecting separated componentsthereof; in particular in combinatorial chemistry; in metabolomics,proteomics or genomics, assay and high throughput analysis applications,typically high sensitivity analyses, separation and/or quantificationstudies and for sample separation for example chromatography orelectrophoresis, in particular column chromatography, capillaryelectrophoresis with real time or post separation analysis; in hospitalor surgery blood tests and other assays, in industrial quality control,in environmental pesticide level monitoring and the like.

The invention is now illustrated in non limiting manner with respect tothe following examples and Figures wherein

Figure A shows elevations of capillary array type vessels and detectionmeans of an optical assembly of the invention

FIG. 1 shows a schematic diagram of apparatus of the invention forparallel capillary absorbance detection

FIG. 2 shows a collimated illumination of rectangular CCD area (26.6×6.7mm) using light output from a 1 mm diameter fused-silica optical fibre(N.A.=0.22) using a cylindrical and spherical fused-silica lens element

FIG. 3 shows the CCD and vessel arrangement of the optical assembly ofthe invention

FIG. 4 shows part of one CCD snapshot showing ˜3 mm of 4 capillaries(100 μm i.d., 194 μm o.d.); the total area imaged is 6.7×26.6 mm. Thecontents of the capillaries are 1. Air, 2. Water, 3 and 4. ink solution

FIG. 5 shows light beam ray tracing diagram according to the inventionshowing light path through a water filled capillary, 100 μm i.d., 194 μmo.d. The dark rays represent the light passing through the water at thecapillary centre and the light rays show the light that passes onlythrough the capillary walls. The dashed line shows the approximateposition of the fibre optic stud surface

FIG. 6 shows light beam tracings not according to the invention showinglight path through an air filled capillary, 100 μm i.d., 194 μm o.d.

FIG. 7 shows light beam tracings according to the invention showinglight path through a water filled capillary, 75 μm i.d., 194 μm o.d.

FIG. 8 shows light beam tracings not according to the invention showinglight path through a water filled capillary, 75 μm i.d., 364 μm o.d.

FIG. 9 shows electropherograms of ˜16 nL 100 microM 4-nitrophenolinjected into each of four parallel 100 μm i.d. capillaries. Capillarylength: 500 mm total, 300 to the detector. Separation voltage: 5000 V.Buffer:sodium phosphate pH 7.5 (15 mM sodium)

FIG. 10 shows electropherograms of 100 microM 4-nitrophenol aftercorrection for cross-talk between capillaries

FIG. 11 shows electropherograms of ˜16 nL 1 microM 4-nitrophenolinjected into each capillary

FIG. 12 shows electropherogram generated by taking the average of thefour traces shown in FIG. 11

FIG. 13 shows peak imaging and variance for a sample of rhodamine 700injected onto a capillary and migrated by CE to the detection zone whichis imaged by laser induced fluorescence with a CCD. The voltage isturned off (at delta t=0) and the peak broadening due to diffusion ismonitored for 1800 s (top). The change in peak variance is plottedagainst time (bottom)

FIG. 14 shows a schematic of a possible arrangement for closed lopfeedback control. The output from a liquid chromatography system ismonitored during a first pass of the area detector. Peak recognition anddecision making software can instruct the switching valve to direct theappropriate fraction for collection or for example to a massspectrometer interface. After the appropriate switch the eluent ismonitored again in a second pass of the detector so that the efficiencyof the switching process can be monitored. An alternative would be tohave more of the outputs from the switching valve passing the detectorfor visualisation

FIG. 15 shows as illumination pattern perpendicular to the capillaryaxis created by light passing through the core of a 75 μm i.d. 194 μmo.d. capillary positioned 260 μm from the detection surface. Plots areshown for the capillary containing water and acetonitrile (at 20° C.,sodium D line); the difference in the illumination pattern would allowthe direct determination of the mobile phase composition during agradient elution separation. Acetonitrile at 44° C. has the samerefractive index as water does at 20° C.; the same approach could beused to determine the mobile phase velocity by applying a heat pulseupstream of the detector

FIG. 16 is a diagram showing an optical assembly clip-on device of theinvention showing the approximate dimensions for absorbance detectionaccording to the invention, such as CE or absorbance. A module device ofthe invention would differ by comprising a capillary with interfacingmeans to insert into a capillary eg from microLC or into massspectrometer.

EXAMPLE 1

The experimental setup is shown in FIG. 1. The output from a 75 W xenonlamp is launched into a single 1 mm diameter fused silica fibre. Thelight output from the fibre is shaped and collimated using cylindricaland spherical fused silica lens elements (FIG. 2) to illuminate therectangular area of the CCD. The CCD chip used is EEV CCD 30-11 and isthermostatted and controlled by a system designed and built by YorkElectronics Centre; it has 1024 by 256 active pixels, each 26 μm square,with a total active area of 6.7×26.6 mm. The CCD chip has a fibre opticstud (faceplate) that protects the CCD surface. The fibre optic stud isnot UV transparent, so a UV phosphor is coated onto the surface of studto make the detector sensitive to a wide wavelength range from NIR tobelow 200 nm. The charge accumulated on the CCD is read out in a seriesof snapshots; to prevent image smearing a light chopper ensures that theCCD is not illuminated during the readout period. An exposure rate of 5Hz is used with a 50% duty cycle (100 ms exposure and 100 ms readouttime). An image of 1024×256 pixels with 14 bit digitisation is obtainedfrom each snapshot.

An ideal arrangement of the capillaries could be to align them parallelto the short axis of the CCD at a spacing of 260 μm (10 pixels percapillary). This arrangement would allow up to 102 capillaries, which isideal to accommodate the number used if sampling from a standard 96 wellplate. The spacing means a gap of 66 μm between each capillary, thiswould allow more reference light reaching the CCD than in the currentarrangement with the capillaries more tightly packed. Better exposurereferencing and lower inter capillary cross talk should result from thisarrangement.

Capillary Imaging

In the demonstration experiment four fused silica capillaries (100 μmi.d., 194 μm o.d. 500 mm long) were placed side by side, approximately200 μm above the CCD and parallel to the long axis of the CCD as shownin FIGS. 1 and 3. The portion of the capillaries imaged was a section273.4 to 300.0 mm from the inlet end. FIG. 4 shows a portion of onesnapshot taken with the four capillaries filled with air, water and thelast two with an ink solution. Excellent referencing is seen for theassembly without any lenses or other optics. It can be seen from thecomparison between capillaries 1 and 2 that the collimated light thatpasses through the capillary core is convergent when the capillary isfilled with water, producing a line that is brighter than thebackground. FIGS. 5 and 6 show in detail how light passes through thewater and air filled capillaries; they show also that in the case of thefilled capillary, there is good separation on the CCD between the lightpassing through the capillary core and that only passing through thecapillary walls. This is confirmed in FIG. 4 where a high contrast isobserved between the images of the water and ink filled capillaries. Itis important to use capillaries with a small o.d.; FIG. 7 shows thatgood results would be obtained using capillaries with the same o.d. of194 μm as used here but with the more commonly used 75 μm i.d. However,the 364 μm o.d. capillary normally used in capillary electrophoresiswould not be suitable; FIG. 8 shows that the larger radius at theair/fused-silica interface does not adequately focus the light havingpassed through the capillary core to spatially separate it from thelight having passed only through the capillary walls. During anexperiment the image from the whole CCD was read into the computer foreach exposure; for this experiment an area of 32×1024 pixels containedthe image of the four capillaries; the remainder was discarded toconserve computer memory. The data was further reduced by addingtogether the pixel values in groups of four down the capillary length togive a total of 32×256 effective pixels for each snapshot; eacheffective pixel has 16 bit resolution and dimensions of 26×104 μm.

Exposure Referencing

Fluctuations in exposure times and in the light source intensity that isin excess of shot noise need to be accounted for in order to get thebest possible performance from the detector. This is usually achieved byusing a double beam arrangement, where a portion of the light from thesource that does not pass through the sample is monitored and used as areference. The ratio of this reference and the light that does passthrough the sample is used to calculate the absorbance. In thisexperiment the light that strikes the CCD having passed only through thecapillary walls is used as the reference. The electrophoresis of4-nitrophenol in its ionic form was used to test the performance of thedetection system. An optical filter with a centre wavelength of 405 nmand bandpass of 10 nm was placed at the input to the fibre optic tomatch the absorbance wavelength of 4-nitrophenol at pH 7.5 (absorbancecoefficient, ε₄₀₅=1700 m² mol⁻¹). To establish which of the image pixelswere to be used as ‘reference pixels’ and which to use as ‘samplepixels’ snapshots with the capillaries filled with water were comparedwith those with the capillaries containing a 1 mM solution of4-nitrophenol. The average pathlength is πd/4=79 μm; this corresponds toan absorbance of 0.13 AU for this solution. The pixels that showed areduction in signal in the presence of the 4-nitrophenol solution thatcorresponded to an absorbance of <0.02 were used as reference pixels,those with >0.1 were used as sample pixels.

Capillary Electrophoresis and Data Processing

A series of parallel capillary electrophoresis experiments were carriedout using a pH 7.5, sodium phosphate buffer (15 mM sodium) on dilutionsof a 4-nitrophenol solution made up in buffer. Injections were performedinserting the inlet end of all the capillaries into the sample solutionthat was held at 2 cm above the level of the buffer in the outlet vialfor 15 s. The volume injected into each capillary by siphoning should beapproximately 16 nL. A voltage of +5 kV was applied to the inlet vialand the electrophoresis was carried out; electroosmotic flow ensuredthat the analyte moved towards the grounded cathode at the outlet. Thesnapshot data was accumulated in the computer RAM during the experimentand saved to disk at the end of a run.

The raw data collected during an experiment was first corrected forfixed pattern noise as described in detail in “A charge coupled devicearray detector for single-wavelength and multi-wavelength ultravioletabsorbance in capillary electrophoresis”, Bergstrom and Goodall, Pokricand Allinson, Anal. Chem. 1999, 71, 4376-4384, and then processed toproduce a set of electropherograms, one for each capillary. This ensuresthat high spatial resolution is maintained, limited only by thedimensions of the effective pixels used, 106 μm in this case.

FIG. 9 shows the four electropherograms obtained using a sample solutionof 100 μM. It is clear from the dips in the base line at timescorresponding to peaks in adjacent capillaries that there is cross talkbetween capillaries. This is mainly caused by some light passing throughthe sample being used as part of the reference, and to a lesser extentthere is some light that after passing through the sample in onecapillary lands on a sample pixel of an adjacent capillary. The extentof both of these effects has been measured by filling each capillary inturn with the sample solution. It has been assumed that the level ofcross talk scales linearly with absorbance and the correction based onthe measurement at this single sample concentration has been applied toall subsequent experiments. The appropriate correction is readily madeas is demonstrated by FIG. 10.

Noise Performance

FIG. 11 shows the four electropherograms obtained by injecting asolution of 1 μM 4-nitrophenol. The peak heights in FIG. 11 correspondto a concentration of ˜0.6 μM, the limit of detection is calculated tobe 0.22 μM based on three times the RMS baseline noise of 9.4 μAU (1 srisetime). The theoretical shot noise limited baseline noise level wascalculated by generating simulated data sets based on the averageexposure patterns and levels found in the experiment and assuming apixel full well capacity of 5×10⁵ electrons. These generated snapshotdata sets were processed identically to the experimental data sets; theRMS shot noise level was found to be 9.0 μAU. This is very close to theobserved value and indicates that the detector is shot noise limited.

The CCD used in this experiment is large enough to accommodate over 30capillaries aligned as the four are here. The sensitivity of detectionfor each of these capillaries would be the same as found in this study(i.e. ˜9 μAU RMS noise with a rise-time of 1 s). Alternatively over 120capillaries could be accommodated if they were aligned parallel to theshort dimension of the CCD; this would result in a reduction in thesignal integration time for each capillary by a factor of four andtherefore an increase in noise level by a factor of {square root}4=2.Capillary electrophoresis with arrays of capillaries obviously hasapplication for high throughput analysis as the number of separationsthat can be carried out simultaneously simply scales with the size ofthe array. However, if the same sample is simultaneously injected intoall the capillaries in the array, as done in this experiment, it is ameans to increase sample loading and therefore dynamic range andconcentration sensitivity. If all of the electropherograms are combinedby taking the arithmetic average after compensation for the slightdifferences in analyte velocities between capillaries, then an increasein signal to noise of {square root}N is achieved, where N is the numberof capillaries. Splitting a large sample load into many capillariesreduces the problems associated with overloading which include poor peakshape induced by electromigration dispersion and large injectionlengths, and nonlinear detector response at high absorbances of highlyabundant components. FIG. 12 shows the result of averaging the fourelectropherograms of FIG. 11. The RMS baseline noise level is 4.7 μAU, areduction by a factor of 2 of the individual traces, this is theimprovement expected for 4 capillaries.

The noise performance measured by combining the signal from fourcapillaries is still at the shot noise limit and is better than theperformance achieved previously in Bergström et al above, where it wassuggested that fluctuations in the spatial distribution of the lampdischarge, which is imaged on the fibre bundle input, would result influctuations in the spatial distribution of the CCD illumination. Thiswould result in a noise level that is in excess of the shot noisebecause separate areas of the CCD monitor the signal and referencelevels. Using a single optical fibre instead of the fibre bundle usedpreviously should to a large degree scramble any spatial inhomogeneitiesof the lamp discharge. Scaling up the experiment to 30 parallelcapillaries should give a combined RMS baseline noise level of 1.7 μAU,corresponding to an on column 4-nitrophenol concentration LOD of 40 nM.

When the analyte concentration is low relative to the concentration ofbackground electrolyte then it should only be the length of theinjection plug and analyte diffusion that contribute significantly tothe measured peak width, assuming that the spatial resolution of thedetector is high. The peak variance caused by analyte diffusion can becalculated, σ²=2Dt=1.2×10⁻⁶ m², where D is the diffusion coefficient(8.1×10⁻¹⁰ m² s⁻¹ for 4-nitrophenol) and t is time. The contribution tothe peak variance from the length of the plug of sample injected isgiven by l_(i) ²/12=0.4×10⁻⁶ m² (where l_(i) is the injection length of2.0 mm). These two contributions give a total standard deviation, σ, of1.3 mm; dividing by the analyte velocity gives σ=3.2 s in units of time.This compares well with a standard deviation of 3.1 s measured byfitting a Gaussian function to the peak in FIG. 12. This is experimentalevidence that the detection method of imaging a 26.6 mm capillarysection and of subsequently combining parallel electropherograms doesnot degrade separation efficiency.

EXAMPLE 2

TABLE 1 Table showing, for vessel with circular outer and inner crosssections, minimum values of ratio of the outer wall to detector distanced and the outer diameter of the vessel o.d. (d/o.d.) in order to obtainspatial separation between core and wall beams. Values are shown as afunction of ratio of inner and outer diameters of the vessel (i.d./o.d.)for a range of solvents and vessel materials. Entries x indicate nospatial separation possible. i.d./o.d. i.d./o.d. i.d./o.d. i.d./o.d.0.20 0.25 0.33 0.50 Water/silica x 2.0 1.0 0.5 Hexane/silica 1.6 1.0 0.60.4 Dichloromethane/silica 0.6 0.5 0.4 0.2 Water/polycarbonate x x 2.10.5 Water/flexible clear 0.5 0.5 0.5 0.4 tubing (Tygon chemfluor 367)

Values of refractive indices used in ray tracing analysis are: silica(1.458), water (1.333), hexane (1.375), dichloromethane (1.424),polycarbonate (1.585), Tygon chemfluor 367 (1.34). Other common reversedphase HPLC solvents, often used in admixture with water, are methanol(1.329) and acetonitrile (1.344): these and their mixtures will givesimilar values of d/o.d to water for the various vessel types. Hexanemay be grouped with two other common normal phase HPLC solvents ofcomparable refractive index, 2-propanol (1.378) and ethyl acetate(1.372). The method used in constructing this Table is also effectivefor determining values for other solvents such as DMSO (1.479).

From the Table, for a known o.d. capillary the required i.d. can bedetermined from one of the possibilities to give a value of d/o.d. andthereby value of d, as described in the description.

EXAMPLE 3 Feedback Control

An example of feedback control is the use in a CE experiment forstopping the sample in the detection window, by turning off the voltagewhen it is detected as having migrated to the centre of the window (seeFIG. 13). After stopping, the subsequent broadening of the sample peakdue to diffusion is monitored as a function of time. Analysis of thechange in peak variance with time enables the diffusion coefficient tobe determined. The hydrodynamic radius of the sample molecule may thenbe calculated using Stokes law. Combination of the diffusion coefficientwith the mobility, obtained from the time taken to reach the windowunder a given field strength, enables the charge on the analyte to bededuced. Any starting peak shape is acceptable, since convolution of thepeak with a Gaussian enables the Gaussian component of the peakbroadening to be extracted. Whilst the example shows peak broadeningmeasured over a period of 30 minutes, data quality is such thatdiffusion coefficients could be measured with good precision in lessthan 3 minutes.

A schematic example of closed-loop feedback control is given in FIG. 14.Here the output from an LC system is monitored during a first pass ofthe area detector. Peak recognition and decision making software is usedto instruct the switching valve to direct the appropriate fraction forcollection or for example to a mass spectrometer interface. After theappropriate switch the eluent is monitored again in a second pass of thedetector, so that the efficiency of the switching process can bemonitored. An alternative is to have more of the outputs of theswitching valve passing the detector for visualisation.

EXAMPLE 4 Uses

The detection method of the invention is used in measuring refractiveindex change. By analysis of the illumination pattern of the core beamat high spatial resolution perpendicular to the capillary axis, thedetector has the ability to monitor refractive index change in thesolvent in the capillary. FIG. 15 shows, for a 75 μm i.d., 194 mm o.d.capillary positioned 260 μm from the detector surface, the change inprofile of the core beam on changing from water (refractive index 1.333)to acetonitrile (refractive index 1.344). With a pixel size of 10 μm,this would allow distinction between the two illumination profiles, andby extension the ability to directly monitor the mobile phasecomposition during a gradient elution separation in water-acetonitrilemixtures. If necessary, magnification in the direction perpendicular tothe capillary axis could be used to increase the resolution in terms ofmobile phase composition. This would be of benefit in HPLC. Sinceacetonitrile at 44° C. has the same refractive index as water does at20° C., application of a heat pulse providing a 25° C. temperature riseup-stream of the detector could be used to determine the mobile phasevelocity in capillary HPLC.

Another use of multicapillary absorbance detection is for rapidmeasurement of pK_(a). Here aliquots of the sample solution are mixedinto a set of buffer solutions covering a range of pH values, typicallyspanning the range 2-12, and all mixtures are then drawn up intoseparate capillaries in the sequence buffer then mixture of buffer plussample. By taking measurements at one or more wavelengths where acid andbase forms have different absorbance, results from all capillaries ofabsorbance as a function of pH may be processed to determine pK_(a)values. Using suitable non- or weakly-absorbing inorganic buffers, thisis applicable for organic or biological compounds containing all commontitratable functional groups, including carboxyl and amine groups, andat concentrations down to 100 micromolar. This is of benefit in forexample the pharmaceutical industry and for high throughput screening.

1-44. (canceled)
 45. An optical assembly comprising a light source, atleast one sample vessel and a detector, the at least one vessel beingpositioned in a light path or paths created between the source and thedetector in manner to enable transmission of light through the vesselwherein the light source is adapted to provide a beam of substantiallycollimated light, the detector comprises a plurality of detectorlocations and the vessel comprises a wall and core of relative shape anddimensions adapted to contain a sample for detection and to define atleast two spatially separated transmitted light paths, a first wall pathwhich enters and exits the vessel walls only, spatially separated from asecond core path which enters and exits the vessel walls andadditionally the vessel core, wherein the spatially separated wall andcore paths are coupled to individual detector locations on the detector,and the detector is an array detector.
 46. Optical assembly of claim 45wherein the assembly defines a central core path and two peripheral wallpaths either side thereof or an annular wall path thereabout. 47.Optical assembly of claim 45 wherein core and wall path beams arespatially close, preferably adjacent, on the array detector,facilitating direct referencing as the ratio of the core beam to thewall beam.
 48. Optical assembly of claim 45 wherein the light sourcecomprises at least one wavelength of light that is absorbed by one ormore absorbing species comprised in the sample for detection, theabsorbance of which is to be detected.
 49. Optical assembly of claim 45wherein light is of wavelength in the range 160 to 1200 nm, preferably180 or 190 to 1200 nm, corresponding to UV, UV-vis to near infra red(NIR).
 50. Optical assembly of claim 45 wherein the at least one samplevessel in the assembly of the invention comprises a cell or conduitwhich is open ended and open based and topped, intended for dynamicsample detection.
 51. Optical assembly of claim 45 wherein the samplevessel is a single cell or one of a plurality of cells in an array; oris a single capillary or one of a plurality of capillaries in amicrocapillary array or a microfabricated channel array.
 52. Opticalassembly of claim 45 characterised by i.d. (inner diameter) (vessel) inthe range 3 micron to 20 mm, o.d. (outer diameter) (vessel) in the range4 micron to 30 mm, refractive index (vessel) in the range 1.3−<1.6,refractive index (sample) in the range 1.3 to in excess of 1.5, ratiod(outer wall to detector distance)/o.d. is 0.5 to 10 and d is in therange 20 micron to 300 mm.
 53. Optical assembly of claim 45 wherein anarray detector comprises a solid state sensing device, preferably a CCD,CID or a CMOS APS.
 54. Optical assembly of claim 45 wherein an arraydetector comprises a CCD, CID or CMOS APS including a surface studcomprising a coating to absorb incident light and reemit at a differentwavelength, to convert UV to visible light, to allow detection by theCCD, CID or CMOS APS wherein the coating is applied directly to the studor to a cover slip interleaved between the stud and vessel, facilitatingrecoating as needed, by replacing the cover slip without need to replacethe stud.
 55. Optical assembly of claim 45 which comprises means forreal-time signal processing for optimum peak detection andparameterisation/characterisation, and means for automatic systemmanagement including closed-loop feedback control of the apparatus andsystems.
 56. Optical assembly of claim 55 in which closed-loop feedbackcontrol means includes means for stopping or slowing the flow followinginitial observation in the detection means to allow sample to reside inthe detector window and give longer times for data acquisition andenhanced signal to noise or to enable fraction collection, or to directa fraction to an analysis means.
 57. Optical assembly of claim 45 whichis a module for use with a column or capillary separating device asknown in the art, wherein the vessel is a capillary or column comprisinginterfacing means at one end for inserting into the outlet of a columnor capillary separating device or along the length thereof, thecapillary or column optionally comprising interfacing means enablinginsertion into the inlet of an analysing means at the other end; or is aclip-on device comprising means for locating about a section of acapillary or column separating device which is of suitable i.d, o.d. andrefractive index as hereinbefore defined and is stripped of any surfacecoating to facilitate the operation of the method of the invention,whereby the stripped capillary or column provides the sample vessel ofthe assembly.
 58. Optical assembly as hereinbefore defined in claim 45for use in the pharmaceutical, biomedical and bioscience, agrochemical,veterinary and materials fields, for detection, analysis,characterisation and quantification of samples contained in a vessel,and optionally further collecting separated components thereof. 59.Apparatus for chemical reaction or synthesis and analysis or for sampleseparation or transport wherein the apparatus comprises the opticalassembly of claim 45 as hereinbefore defined in which the chemicalreaction vessel itself is cylindrical and the reaction monitored inbatch flow mode as a function of time, and feedback control used to haltreaction or in which the reaction vessel is tubular and used incontinuous flow mode.
 60. Method for detection of light transmittedthrough at least one sample contained within the core of at least onesample vessel of an optical assembly as hereinbefore defined in claim45, comprising illuminating the vessel with a substantially collimatedlight source or sources and detecting transmitted light in an arraydetector, wherein transmitted light is spatially separated into at leasttwo light paths, a wall path which has passed through the vessel wallsonly, spatially separated from a core path which has passed through thewalls and core, wherein the spatially separated light beams are coupledto individual detection locations on the array detector.
 61. Method ofclaim 60 wherein a sample includes one or a plurality of analytes whichit is desired to detect in the course of a chemical reaction generatingor consuming a species as analyte.
 62. Method of claim 60 whichadditionally comprises selecting a sample for analysis, determiningindividual wavelengths at which absorption by desired sample componentsis strongest, checking refractive index of the sample in order to selecta suitable sample vessel which when containing the sample and whenilluminated will generate spatially separated beams as hereinbeforedefined or selecting a suitable combination of optical components andfilters and a suitable vessel to detect an array separation to couplespatially separated beams to independent locations on the detectorarray.
 63. Method of claim 60 in which sample is introduced into the atleast one sample vessel by injection, loop injection, pipette,hydrostatic, or electrokinetic injection and is removed from the vesselby injection, electrospray or interface for discard or to a furthervessel for storage or to a down stream identification means.
 64. Methodof claim 60 which comprises referencing the light detected by thedetection means by means of exposure referencing wherein the ratio ofthe core beam intensity to the wall beam intensity gives a value for thesample intensity at each location with elimination of excess or flickernoise due to light source fluctuation.